Introduction
Sturgeons (Acipenseriformes ) are ecologically and economically significant fish facing severe population declines, necessitating advanced conservation strategies. Sperm cryopreservation is critical for ex-situ genetic preservation and enhancing aquaculture productivity through artificial fertilization. Developing effective cryopreservation protocols requires understanding the unique characteristics of sturgeon spermatozoa, particularly their dependence on potassium ions (K+), which are critical for inhibiting motility. We hypothesized that a hypotonic extender containing only 1 mM KCl could effectively inhibit sperm motility and support successful cryopreservation, offering an alternative approach compared to standard sturgeon protocols.
Materials and methods
This study evaluated sperm cryopreservation protocols across sterlet ( Acipenser ruthenus), Siberian ( A. baerii), Russian ( A. gueldenstaedtii), and stellate (A. stellatus ) sturgeons, comparing a novel cryomedium (1 mM KCl , 11% methanol, 2.5% ethylene glycol) against a traditional cryomedium. Two cryopreservation formats were tested: 0.5 ml straws with rapid cooling on a polystyrene raft in a Styrofoam box, and 4.5 ml and 10 ml cryotubes with slow, controlled cooling in a programmable freezer. Post-thaw motility, spermatozoa volume changes during equilibration, fertilization success, and embryogenesis were assessed.
Results
The hypotonic cryomedium preserved high post-thaw motility: Siberian sturgeon achieved 46±6% (0.5 ml straws and 4.5 ml cryotubes); Russian sturgeon, 31±10% (0.5 ml straws) and 40±12% (10 ml cryotubes); and stellate sturgeon, 46±10% (0.5 ml straws), 39±8% (4.5 ml cryotubes), and 44±11% (10 ml cryotubes). Fertilization and hatching rates were robust, with Siberian sturgeon reaching 81±16% and 79±15% (0.5 ml straws), and 84±6% and 82±7% (4.5 ml cryotubes), and Russian sturgeon achieving 60±20% and 57±20% (0.5 ml straws), and 62±11% and 48±13% (10 ml cryotubes). Ethylene glycol induced initial sperm cell shrinkage followed by volume normalization, unlike methanol, which caused minimal volume changes. Although curvilinear velocity (VCL) was significantly reduced in the hypotonic cryomedium compared to fresh sperm and traditional cryomedium (p<0.05), fertilization success remained unaffected, and embryos showed no significant morphological abnormalities.
Conclusion
This study confirms the effectiveness of the 1 mM KCl -based hypotonic cryomedium for cryopreserving sperm across multiple sturgeon species, and sample volumes, supporting both small-scale (0.5 ml straws) and large-scale (4.5–10 ml cryotubes) cryopreservation without compromising reproductive outcomes. This protocol offers a versatile, scalable approach for sturgeon sperm cryopreservation, advancing conservation and aquaculture practices by facilitating large-scale breeding.